Hepatocellular carcinoma (HCC), the most prevalent liver cancer, ranks third in cancer-related mortality because of ineffective therapy. HCC almost always arises due to complication of underlying liver disease, e.g. hepatitis, fibrosis and cirrhosis. Identification of cellular and molecular entities that directly or indirectly contributeto HCC development will be central to achieve the goal of developing effective therapy. One such molecule that is critical to the maintenance of normal liver function is miR-122, the most abundant liver-specific microRNA. Down regulation of this small (22-nucleotide) non-coding regulatory RNA is associated with poor prognosis, tumor recurrence and metastasis. We found that in mice, disruption of miR-122 action in liver results in development of highly penetrant HCC. These liver-specific miR-122 knockout (LKO) mice spontaneously develop hepatitis, fibrosis and HCC with lung metastasis. More importantly, others and we found that miR-122 delivery via the viral vector (AAV8) or liposomal nanoparticles inhibited HCC development in different mouse models, suggesting a therapeutic potential of miR-122 in HCC patients. We now propose to use our novel mouse model to delineate the underlying mechanism of hepatitis, fibrosis and hepatocarcinogenesis, and to develop effective therapeutics for liver cancer. To this end, we propose the following aims. Aim 1 will (a) validate functional targets of miR-122 that are identified in the wild type liver transcriptome by unbiased HITS-CLIP and RNA-seq approach using miR-122KO livers as a negative control, and (b) investigate anti-correlation between miR-122 and these targets in primary human HCC. We will focus on the targets that promote tumorigenesis at different stages. Aim 2 will test therapeutic efficacy of anti- inflammatory (Ccl2 neutralizing Ab) and anti-fibrotic (Ctgf neutralizing Ab) agents in combination with miR-122 delivered using HCC-targeted liposomal nanoparticles. We will focus on the Ccl2-Ccr2 axis since our supporting data showed that blocking this axis reduced inflammation, fibrosis and tumor burden in these mice, and on Ctgf because it is a direct target of miR-122 identified by HITS-CLIP. Studies proposed here will elucidate the mechanism of suppression of hepatic fibrosis and hepatocarcinogenesis by blocking functions of Ccl2 and Ctgf in combination with miR-122. Aim 3 will decipher the role of the cellular oncogene c-Myc and its target glutaminase 1 (Gls1) that deamidates glutamine to glutamate, a key energy source to cancer cells, in HCC development in LKO mice using clinically relevant c-Myc and Gls1 inhibitors. This aim draws on our finding that blocking c-Myc gene expression, using the small molecule drug JQ1, inhibited growth, clonogenic survival and promoted apoptosis of cultured human HCC cells. Further, using isotopologue studies, we found that LKO livers and tumors show increased conversion of glutamine to glutamate that correlated with an increase in c-Myc-regulated Gls1, an enzyme facilitating utilization of glutamine as an energy source. Collectively, the proposed studies will generate pre-clinical results that will be key for designing future clinical trials with one or mor of the above chemotherapeutic agents in combination with miR-122 for treatment of patients with HCC.